Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

RELATED APPLICATIONS

The present application claims priority to provisional application U.S.Ser. No. (60/207,281), filed May 30, 2000.

FIELD OF THE INVENTION

The present invention is in the field of kinase proteins that arerelated to the calmodulin-binding kinase subfamily, recombinant DNAmolecules, and protein production. The present invention specificallyprovides novel peptides and proteins that effect protein phosphorylationand nucleic acid molecules encoding such peptide and protein molecules,all of which are useful in the development of human therapeutics anddiagnostic compositions and methods.

BACKGROUND OF THE INVENTION Protein Kinases

Kinases regulate many different cell proliferation, differentiation, andsignaling processes by adding phosphate groups to proteins. Uncontrolledsignaling has been implicated in a variety of disease conditionsincluding inflammation, cancer, arteriosclerosis, and psoriasis.Reversible protein phosphorylation is the main strategy for controllingactivities of eukaryotic cells. It is estimated that more than 1000 ofthe 10,000 proteins active in a typical mammalian cell arephosphorylated. The high energy phosphate, which drives activation, isgenerally transferred from adenosine triphosphate molecules (ATP) to aparticular protein by protein kinases and removed from that protein byprotein phosphatases. Phosphorylation occurs in response toextracellular signals (hormones, neurotransmitters, growth anddifferentiation factors, etc), cell cycle checkpoints, and environmentalor nutritional stresses and is roughly analogous to turning on amolecular switch. When the switch goes on, the appropriate proteinkinase activates a metabolic enzyme, regulatory protein, receptor,cytoskeletal protein, ion channel or pump, or transcription factor.

The kinases comprise the largest known protein group, a superfamily ofenzymes with widely varied functions and specificities. They are usuallynamed after their substrate, their regulatory molecules, or some aspectof a mutant phenotype. With regard to substrates, the protein kinasesmay be roughly divided into two groups; those that phosphorylatetyrosine residues (protein tyrosine kinases, PTK) and those thatphosphorylate serine or threonine residues (serine/threonine kinases,STK). A few protein kinases have dual specificity and phosphorylatethreonine and tyrosine residues. Almost all kinases contain a similar250-300 amino acid catalytic domain. The N-terminal domain, whichcontains subdomains I-IV, generally folds into a two-lobed structure,which binds and orients the ATP (or GTP) donor molecule. The larger Cterminal lobe, which contains subdomains VI A-XI, binds the proteinsubstrate and carries out the transfer of the gamma phosphate from ATPto the hydroxyl group of a serine, threonine, or tyrosine residue.Subdomain V spans the two lobes.

The kinases may be categorized into families by the different amino acidsequences (generally between 5 and 100 residues) located on either sideof, or inserted into loops of, the kinase domain. These added amino acidsequences allow the regulation of each kinase as it recognizes andinteracts with its target protein. The primary structure of the kinasedomains is conserved and can be further subdivided into 11 subdomains.Each of the 11 subdomains contains specific residues and motifs orpatterns of amino acids that are characteristic of that subdomain andare highly conserved (Hardie, G. and Hanks, S. (1995) The Protein KinaseFacts Books, Vol 1:7-20 Academic Press, San Diego, Calif.).

The second messenger dependent protein kinases primarily mediate theeffects of second messengers such as cyclic AMP (cAMP), cyclic GMP,inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate,cyclic-ADPribose, arachidonic acid, diacylglycerol andcalcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) areimportant members of the STK family. Cyclic-AMP is an intracellularmediator of hormone action in all prokaryotic and animal cells that havebeen studied. Such hormone-induced cellular responses include thyroidhormone secretion, cortisol secretion, progesterone secretion, glycogenbreakdown, bone resorption, and regulation of heart rate and force ofheart muscle contraction. PKA is found in all animal cells and isthought to account for the effects of cyclic-AMP in most of these cells.Altered PKA expression is implicated in a variety of disorders anddiseases including cancer, thyroid disorders, diabetes, atherosclerosis,and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison'sPrinciples of Internal Medicine, McGraw-Hill, New York, N.Y., pp.416-431, 1887).

Another ligand-activated protein kinase is 5′-AMP-activated proteinkinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81).Mammalian AMPK is a regulator of fatty acid and sterol synthesis throughphosphorylation of the enzymes acetyl-CoA carboxylase andhydroxymethylglutaryl-CoA reductase and mediates responses of thesepathways to cellular stresses such as heat shock and depletion ofglucose and ATP. AMPK is a heterotrimeric complex comprised of acatalytic alpha subunit and two non-catalytic beta and gamma subunitsthat are believed to regulate the activity of the alpha subunit.Subunits of AMPK have a much wider distribution in nonlipogenic tissuessuch as brain, heart, spleen, and lung than expected. This distributionsuggests that its role may extend beyond regulation of lipid metabolismalone.

The mitogen-activated protein kinases (MAP) are also members of the STKfamily. MP kinases also regulate intracellular signaling pathways. Theymediate signal transduction from the cell surface to the nucleus viaphosphorylation cascades. Several subgroups have been identified, andeach manifests different substrate specificities and responds todistinct extracellular stimuli (Egan, S. E. and Weinberg, R. A. (1993)Nature 365:781-783). MAP kinase signaling pathways are present inmammalian cells as well as in yeast. The extracellular stimuli thatactivate mammalian pathways include epidermal growth factor (EGF),ultraviolet light, hyperosmolar medium, heat shock, endotoxiclipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumornecrosis factor (TNF) and interleukin-I (IL-1).

PRK (proliferation-related kinase) is a serum/cytokine inducible STKthat is involved in regulation of the cell cycle and cell proliferationin human megakaroytic cells (Li, B. et al. (1996) J. Biol. Chem.271:19402-8). PRK is related to the polo (derived from humans polo gene)family of STKs implicated in cell division. PRK is downregulated in lungtumor tissue and may be a proto-oncogene whose deregulated expression innormal tissue leads to oncogenic transformation. Altered MAP kinaseexpression is implicated in a variety of disease conditions includingcancer, inflammation, immune disorders, and disorders affecting growthand development.

The cyclin-dependent protein kinases (CDKs) are another group of STKsthat control the progression of cells through the cell cycle. Cyclinsare small regulatory proteins that act by binding to and activating CDKsthat then trigger various phases of the cell cycle by phosphorylatingand activating selected proteins involved in the mitotic process. CDKsare unique in that they require multiple inputs to become activated. Inaddition to the binding of cyclin, CDK activation requires thephosphorylation of a specific threonine residue and thedephosphorylation of a specific tyrosine residue.

Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosineresidues on their target proteins and may be divided into transmembrane,receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembraneprotein-tyrosine kinases are receptors for most growth factors. Bindingof growth factor to the receptor activates the transfer of a phosphategroup from ATP to selected tyrosine side chains of the receptor andother specific proteins. Growth factors (GF) associated with receptorPTKs include; epidermal GF, platelet-derived GF, fibroblast GF,hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascularendothelial GF, and macrophage colony stimulating factor.

Non-receptor PTKs lack transmembrane regions and, instead, formcomplexes with the intracellular regions of cell surface receptors. Suchreceptors that function through non-receptor PTKs include those forcytokines, hormones (growth hormone and prolactin) and antigen-specificreceptors on T and B lymphocytes.

Many of these PTKs were first identified as the products of mutantoncogenes in cancer cells where their activation was no longer subjectto normal cellular controls. In fact, about one third of the knownoncogenes encode PTKs, and it is well known that cellular transformation(oncogenesis) is often accompanied by increased tyrosine phosphorylationactivity (Carbonneau H and Tonks N K (1992) Annu. Rev. Cell. Biol.8:463-93). Regulation of PTK activity may therefore be an importantstrategy in controlling some types of cancer.

Calmodulin-Binding Kinases

Calmodulin-binding protein kinases, including calcium/calmodulin (CaM)dependent protein kinases, are members of the STK family. Calmodulin isa calcium receptor that mediates many calcium regulated processes bybinding to target proteins in response to the binding of calcium. Theprinciple target protein in these processes is CaM dependent proteinkinases. CaM-kinases are involved in regulation of smooth musclecontraction (MLC kinase), glycogen breakdown (phosphorylase kinase), andneurotransmission (CaM kinase I and CaM kinase II). CaM kinase Iphosphorylates a variety of substrates including the neurotransmitterrelated proteins synapsin I and II, the gene transcription regulator,CREB, and the cystic fibrosis conductance regulator protein, CFTR(Haribabu, B. et al. (1995) EMBO Journal 14:3679-86). CaM II kinase alsophosphorylates synapsin at different sites, and controls the synthesisof catecholamines in the brain through phosphorylation and activation oftyrosine hydroxylase. Many of the CaM kinases are activated byphosphorylation in addition to binding to CaM. The kinase mayautophosphorylate itself, or be phosphorylated by another kinase as partof a “kinase cascade”.

Calmodulin is a major Ca(2+)-binding protein in the brain, where itmodulates numerous Ca(2+)-dependent enzymes and cellular functions.Ca2+/calmodulindependent protein kinase II (CaMKII) is particularlyimportant in the brain and is involved in a variety of neuronalfunctions (Sola et al., Prog Neurobiol 1999 June;58(3):207-32), such aspostsynaptic responses (such as long-term potentiation),neurotransmitter synthesis and exocytosis, cytoskeletal interactions andgene transcription (Colbran, Neurochem Int December 1992; 21(4):469-97).Ca2+ and calmodulin antagonists inhibit seizures induced by convulsantagents, indicating that the Ca2+/calmodulin signaling system plays animportant role in the onset of seizures. Changes in CaMKII expressionhas been observed following seizures and, furthermore, expression ofcalmodulin and CaMKII in microglial cells in the brain increasesfollowing seizures (Sola et al., Prog Neurobiol June 1999;58(3):207-32). CaMKII levels are also altered in pathological statessuch as Alzheimer's disease and ischemia (Colbran, Neurochem IntDecember 1992; 21(4):469-97), suggesting a role of CaMKII in thesedisorders.

Calmodulin is also important for regulating the plasma membrane calciumpump, which transports Ca2+ out of cells. The pump is inactive in theabsence of calmodulin, but is activated by calmodulin binding (Pennistonet al., J Membr Biol September 1992 15;165(2):101-9).

Kinase proteins, particularly members of the calnodulin-binding kinasesubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of kinase proteins. The present invention advances the stateof the art by providing previously unidentified human kinase proteinsthat have homology to members of the calmodulin-binding kinasesubfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human kinase peptides and proteins that are related tothe calmodulinbinding kinase subfamily, as well as allelic variants andother mammalian orthologs thereof. These unique peptide sequences, andnucleic acid sequences that encode these peptides, can be used as modelsfor the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate kinase activity incells and tissues that express the kinase. Experimental data as providedin FIG. 1 indicates expression in humans in the brain (including infantand adult brain), lung, and hippocampus.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodesthe kinase protein of the present invention. (SEQ ID NO:1) In addition,structure and functional information is provided, such as ATG start,stop and tissue distribution, where available, that allows one toreadily determine specific uses of inventions based on this molecularsequence. Experimental data as provided in FIG. 1 indicates expressionin humans in the brain (including infant and adult brain), lung, andhippocampus.

FIG. 2 provides the predicted amino acid sequence of the kinase of thepresent invention. (SEQ ID NO:2) In addition structure and functionalinformation such as protein family, function, and modification sites isprovided where available, allowing one to readily determine specificuses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding the kinaseprotein of the present invention. (SEQ ID NO:3) In addition structureand functional information, such as intron/exon structure, promoterlocation, etc., is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.As illustrated in FIG. 3, SNPs were identified at nine differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a kinase protein or part of a kinase protein and arerelated to the calmodulin-binding kinase subfamily. Utilizing thesesequences, additional genomic sequences were assembled and transcriptand/or cDNA sequences were isolated and characterized. Based on thisanalysis, the present invention provides amino acid sequences of humankinase peptides and proteins that are related to the calmodulinbindingkinase subfamily, nucleic acid sequences in the form of transcriptsequences, cDNA sequences and/or genomic sequences that encode thesekinase peptides and proteins, nucleic acid variation (allelicinformation), tissue distribution of expression, and information aboutthe closest art known protein/peptide/domain that has structural orsequence homology to the kinase of the present invention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known kinase proteins of thecalmodulin-binding kinase subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including infant and adult brain), lung, and hippocampus.The art has clearly established the commercial importance of members ofthis family of proteins and proteins that have expression patternssimilar to that of the present gene. Some of the more specific featuresof the peptides of the present invention, and the uses thereof, aredescribed herein, particularly in the Background of the Invention and inthe annotation provided in the Figures, and/or are known within the artfor each of the known calmodulin-binding family or subfamily of kinaseproteins.

Specific Embodiments Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thekinase family of proteins and are related to the calmodulin-bindingkinase subfamily (protein sequences are provided in FIG. 2,transcript/cDNA sequences are provided in FIG. 1 and genomic sequencesare provided in FIG. 3). The peptide sequences provided in FIG. 2, aswell as the obvious variants described herein, particularly allelicvariants as identified herein and using the information in FIG. 3, willbe referred herein as the kinase peptides of the present invention,kinase peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the kinase peptides disclosed in the FIG. 2, (encoded bythe nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thekinase peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

The isolated kinase peptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including infant and adult brain), lung, and hippocampus.For example, a nucleic acid molecule encoding the kinase peptide iscloned into an expression vector, the expression vector introduced intoa host cell and the protein expressed in the host cell. The protein canthen be isolated from the cells by an appropriate purification schemeusing standard protein purification techniques. Many of these techniquesare described in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ IDNO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the kinase peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

The kinase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a kinase peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the kinase peptide. “Operatively linked”indicates that the kinase peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the kinase peptide.

In some uses, the fusion protein does not affect the activity of thekinase peptide per se. For example, the fusion protein can include, butis not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant kinase peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A kinase peptide-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the kinase peptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the kinase peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM 120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thekinase peptides of the present invention as well as being encoded by thesame genetic locus as the kinase peptide provided herein. As indicatedby the data presented in FIG. 3, the gene encoding the novel humankinase provided by the present invention is located on public BACAC068701, which is known to be mapped to chromosome 3.

Allelic variants of a kinase peptide can readily be identified as beinga human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the kinase peptide as well asbeing encoded by the same genetic locus as the kinase peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. As indicated by the data presented in FIG. 3, thegene encoding the novel human kinase provided by the present inventionis located on public BAC AC068701, which is known to be mapped tochromosome 3. As used herein, two proteins (or a region of the proteins)have significant homology when the amino acid sequences are typically atleast about 70-80%, 80-90%, and more typically at least about 90-95% ormore homologous. A significantly homologous amino acid sequence,according to the present invention, will be encoded by a nucleic acidsequence that will hybridize to a kinase peptide encoding nucleic acidmolecule under stringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in the geneencoding the kinase protein of the present invention. SNPs wereidentified at nine different nucleotide positions, including sixpositions outside the ORF. SNPs outside the ORF may affectcontrol/regulatory elements.

Paralogs of a kinase peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the kinase peptide, as being encoded by a gene from humans, and ashaving similar activity or function. Two proteins will typically beconsidered paralogs when the amino acid sequences are typically at leastabout 60% or greater, and more typically at least about 70% or greaterhomology through a given region or domain. Such paralogs will be encodedby a nucleic acid sequence that will hybridize to a kinase peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a kinase peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the kinase peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from mammals, preferablyprimates, for the development of human therapeutic targets and agents.Such orthologs will be encoded by a nucleic acid sequence that willhybridize to a kinase peptide encoding nucleic acid molecule undermoderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the kinase peptides of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the kinase peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a kinase peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Be;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

Variant kinase peptides can be fully functional or can lack function inone or more activities, e.g. ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variation orvariation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as kinase activity or in assays such as an in vitro proliferativeactivity. Sites that are critical for binding partner/substrate bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance or photoaffinity labeling (Smith et al., J.Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312(1992)).

The present invention further provides fragments of the kinase peptides,in addition to proteins and peptides that comprise and consist of suchfragments, particularly those comprising the residues identified in FIG.2. The fragments to which the invention pertains, however, are not to beconstrued as encompassing fragments that may be disclosed publicly priorto the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a kinase peptide. Such fragments canbe chosen based on the ability to retain one or more of the biologicalactivities of the kinase peptide or could be chosen for the ability toperform a function, e.g. bind a substrate or act as an immunogen.Particularly important fragments are biologically active fragments,peptides that are, for example, about 8 or more amino acids in length.Such fragments will typically comprise a domain or motif of the kinasepeptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in kinase peptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art (someof these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the kinase peptides of the present invention also encompassderivatives or analogs in which a substituted amino acid residue is notone encoded by the genetic code, in which a substituent group isincluded, in which the mature kinase peptide is fused with anothercompound, such as a compound to increase the half-life of the kinasepeptide (for example, polyethylene glycol), or in which the additionalamino acids are fused to the mature kinase peptide, such as a leader orsecretory sequence or a sequence for purification of the mature kinasepeptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theFigures; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a kinase-effectorprotein interaction or kinase-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, kinases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the kinase. Experimental data as providedin FIG. 1 indicates that kinase proteins of the present invention areexpressed in humans in the brain (including infant and adult brain),lung, and hippocampus. Specifically, a virtual northern blot showsexpression in the brain (adult and infant) and lung. In addition,PCR-based tissue screening panels indicate expression in the infantbrain and hippocampus. A large percentage of pharmaceutical agents arebeing developed that modulate the activity of kinase proteins,particularly members of the calmodulin-binding subfamily (see Backgroundof the Invention). The structural and functional information provided inthe Background and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in humans in the brain (including infantand adult brain), lung, and hippocampus. Such uses can readily bedetermined using the information provided herein, that which is known inthe art, and routine experimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to kinases that are related to members ofthe calmodulin-binding subfamily. Such assays involve any of the knownkinase functions or activities or properties useful for diagnosis andtreatment of kinase-related conditions that are specific for thesubfamily of kinases that the one of the present invention belongs to,particularly in cells and tissues that express the kinase. Experimentaldata as provided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in humans in the brain (including infant andadult brain), lung, and hippocampus. Specifically, a virtual northernblot shows expression in the brain (adult and infant) and lung. Inaddition, PCR-based tissue screening panels indicate expression in theinfant brain and hippocampus.

The proteins of the present invention are also useful in drug screeningassays, in cellbased or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the kinase, as a biopsy orexpanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in the brain (including infant and adultbrain), lung, and hippocampus. In an alternate embodiment, cell-basedassays involve recombinant host cells expressing the kinase protein.

The polypeptides can be used to identify compounds that modulate kinaseactivity of the protein in its natural state or an altered form thatcauses a specific disease or pathology associated with the kinase. Boththe kinases of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the kinase. These compounds can befurther screened against a functional kinase to determine the effect ofthe compound on the kinase activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the kinase to a desired degree.

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between thekinase protein and a molecule that normally interacts with the kinaseprotein, e.g. a substrate or a component of the signal pathway that thekinase protein normally interacts (for example, another kinase). Suchassays typically include the steps of combining the kinase protein witha candidate compound under conditions that allow the kinase protein, orfragment, to interact with the target molecule, and to detect theformation of a complex between the protein and the target or to detectthe biochemical consequence of the interaction with the kinase proteinand the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantkinases or appropriate fragments containing mutations that affect kinasefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) kinase activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate kinase activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the kinase proteindependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the kinasecan be used as an endpoint assay. These include all of the biochemicalor biochemical/biological events described herein, in the referencescited herein, incorporated by reference for these endpoint assaytargets, and other functions known to those of ordinary skill in the artor that can be readily identified using the information provided in theFigures, particularly FIG. 2. Specifically, a biological function of acell or tissues that expresses the kinase can be assayed. Experimentaldata as provided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in humans in the brain (including infant andadult brain), lung, and hippocampus. Specifically, a virtual northernblot shows expression in the brain (adult and infant) and lung. Inaddition, PCR-based tissue screening panels indicate expression in theinfant brain and hippocampus.

Binding and/or activating compounds can also be screened by usingchimeric kinase proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native kinase. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the kinase is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the kinase (e.g. binding partners and/or ligands). Thus, a compoundis exposed to a kinase polypeptide under conditions that allow thecompound to bind or to otherwise interact with the polypeptide. Solublekinase polypeptide is also added to the mixture. If the test compoundinteracts with the soluble kinase polypeptide, it decreases the amountof complex formed or activity from the kinase target. This type of assayis particularly useful in cases in which compounds are sought thatinteract with specific regions of the kinase. Thus, the solublepolypeptide that competes with the target kinase region is designed tocontain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the kinase protein, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofkinase-binding protein found in the bead fraction quantitated from thegel using standard electrophoretic techniques. For example, either thepolypeptide or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies reactive with the protein but whichdo not interfere with binding of the protein to its target molecule canbe derivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of a kinase-binding proteinand a candidate compound are incubated in the kinase protein-presentingwells and the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the kinase protein targetmolecule, or which are reactive with kinase protein and compete with thetarget molecule, as well as enzyme-linked assays which rely on detectingan enzymatic activity associated with the target molecule.

Agents that modulate one of the kinases of the present invention can beidentified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of kinase protein activity identified according to these drugscreening assays can be used to treat a subject with a disorder mediatedby the kinase pathway, by treating cells or tissues that express thekinase. Experimental data as provided in FIG. 1 indicates expression inhumans in the brain (including infant and adult brain), lung, andhippocampus. These methods of treatment include the steps ofadministering a modulator of kinase activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

In yet another aspect of the invention, the kinase proteins can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the kinase and are involved in kinase activity.Such kinase-binding proteins are also likely to be involved in thepropagation of signals by the kinase proteins or kinase targets as, forexample, downstream elements of a kinase-mediated signaling pathway.Alternatively, such kinase-binding proteins are likely to be kinaseinhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a kinase proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming akinase-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the kinase protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a kinase-modulating agent, an antisense kinasenucleic acid molecule, a kinase-specific antibody, or a kinase-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

The kinase proteins of the present invention are also useful to providea target for diagnosing a disease or predisposition to disease mediatedby the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in the brain (including infant and adultbrain), lung, and hippocampus. The method involves contacting abiological sample with a compound capable of interacting with the kinaseprotein such that the interaction can be detected. Such an assay can beprovided in a single detection format or a multi-detection format suchas an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered kinase activity in cell-based orcell-free assay, alteration in substrate or antibodybinding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the kinase protein in which one ormore of the kinase functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and kinase activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including infant and adult brain), lung, and hippocampus.Accordingly, methods for treatment include the use of the kinase proteinor fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe kinase proteins. Antibodies can be prepared from any region of thepeptide as described herein. However, preferred regions will includethose involved in function/activity and/or kinase/binding partnerinteraction. FIG. 2 can be used to identify particularly importantregions while sequence alignment can be used to identify conserved andunique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidinpbiotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates thatkinase proteins of the present invention are expressed in humans in thebrain (including infant and adult brain), lung, and hippocampus.Specifically, a virtual northern blot shows expression in the brain(adult and infant) and lung. In addition, PCR-based tissue screeningpanels indicate expression in the infant brain and hippocampus. Further,such antibodies can be used to detect protein in situ, in vitro, or in acell lysate or supernatant in order to evaluate the abundance andpattern of expression. Also, such antibodies can be used to assessabnormal tissue distribution or abnormal expression during developmentor progression of a biological condition. Antibody detection ofcirculating fragments of the fall length protein can be used to identifyturnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the brain (including infant and adult brain),lung, and hippocampus. If a disorder is characterized by a specificmutation in the protein, antibodies specific for this mutant protein canbe used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including infant and adult brain), lung, and hippocampus.The diagnostic uses can be applied, not only in genetic testing, butalso in monitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting expression level or the presence ofaberrant sequence and aberrant tissue distribution or developmentalexpression, antibodies directed against the protein or relevantfragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in humans in the brain(including infant and adult brain), lung, and hippocampus. Thus, where aspecific protein has been correlated with expression in a specifictissue, antibodies that are specific for this protein can be used toidentify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the kinase peptide to a binding partnersuch as a substrate. These uses can also be applied in a therapeuticcontext in which treatment involves inhibiting the protein's function.An antibody can be used, for example, to block binding, thus modulating(agonizing or antagonizing) the peptides activity. Antibodies can beprepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornuleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a kinase peptide or protein of the present invention (cDNA,transcript and genomic sequence). Such nucleic acid molecules willconsist of, consist essentially of, or comprise a nucleotide sequencethat encodes one of the kinase peptides of the present invention, anallelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule consists of a nucleotide sequence when thenucleotide sequence is the complete nucleotide sequence of the nucleicacid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule consists essentially of a nucleotidesequence when such a nucleotide sequence is present with only a fewadditional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule comprises a nucleotide sequence when thenucleotide sequence is at least part of the final nucleotide sequence ofthe nucleic acid molecule. In such a fashion, the nucleic acid moleculecan be only the nucleotide sequence or have additional nucleic acidresidues, such as nucleic acid residues that are naturally associatedwith it or heterologous nucleotide sequences. Such a nucleic acidmolecule can have a few additional nucleotides or can comprises severalhundred or more additional nucleotides. A brief description of howvarious types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the kinase peptide alone, thesequence encoding the mature peptide and additional coding sequences,such as a leader or secretory sequence (e.g., a prepro or pro-proteinsequence), the sequence encoding the mature peptide, with or without theadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut non-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the nucleic acid moleculemay be fused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the kinase proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnonconservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in FIGS. 1 and 3. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the gene encoding the novel human kinaseprovided by the present invention is located on public BAC AC068701,which is known to be mapped to chromosome 3.

FIG. 3 provides information on SNPs that have been found in the geneencoding the kinase protein of the present invention. SNPs wereidentified at nine different nucleotide positions, including sixpositions outside the ORF. SNPs outside the ORF may affectcontrol/regulatory elements.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2. Asillustrated in FIG. 3, SNPs were identified at nine different nucleotidepositions.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. As indicated by the data presented in FIG. 3, thegene encoding the novel human kinase provided by the present inventionis located on public BAC AC068701, which is known to be mapped tochromosome 3.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates thatkinase proteins of the present invention are expressed in humans in thebrain (including infant and adult brain), lung, and hippocampus.Specifically, a virtual northern blot shows expression in the brain(adult and infant) and lung. In addition, PCR-based tissue screeningpanels indicate expression in the infant brain and hippocampus.Accordingly, the probes can be used to detect the presence of, or todetermine levels of, a specific nucleic acid molecule in cells, tissues,and in organisms. The nucleic acid whose level is determined can be DNAor RNA. Accordingly, probes corresponding to the peptides describedherein can be used to assess expression and/or gene copy number in agiven cell, tissue, or organism. These uses are relevant for diagnosisof disorders involving an increase or decrease in kinase proteinexpression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a kinase protein, such as by measuring alevel of a kinase-encoding nucleic acid in a sample of cells from asubject e.g., mRNA or genomic DNA, or determining if a kinase gene hasbeen mutated. Experimental data as provided in FIG. 1 indicates thatkinase proteins of the present invention are expressed in humans in thebrain (including infant and adult brain), lung, and hippocampus.Specifically, a virtual northern blot shows expression in the brain(adult and infant) and lung. In addition, PCR-based tissue screeningpanels indicate expression in the infant brain and hippocampus.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate kinase nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe kinase gene, particularly biological and pathological processes thatare mediated by the kinase in cells and tissues that express it.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including infant and adult brain), lung, and hippocampus.The method typically includes assaying the ability of the compound tomodulate the expression of the kinase nucleic acid and thus identifyinga compound that can be used to treat a disorder characterized byundesired kinase nucleic acid expression. The assays can be performed incell-based and cell-free systems. Cell-based assays include cellsnaturally expressing the kinase nucleic acid or recombinant cellsgenetically engineered to express specific nucleic acid sequences.

The assay for kinase nucleic acid expression can involve direct assay ofnucleic acid levels, such as mRNA levels, or on collateral compoundsinvolved in the signal pathway. Further, the expression of genes thatare up- or down-regulated in response to the kinase protein signalpathway can also be assayed. In this embodiment the regulatory regionsof these genes can be operably linked to a reporter gene such asluciferase.

Thus, modulators of kinase gene expression can be identified in a methodwherein a cell is contacted with a candidate compound and the expressionof mRNA determined. The level of expression of kinase mRNA in thepresence of the candidate compound is compared to the level ofexpression of kinase mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate kinase nucleic acid expression in cells andtissues that express the kinase. Experimental data as provided in FIG. 1indicates that kinase proteins of the present invention are expressed inhumans in the brain (including infant and adult brain), lung, andhippocampus. Specifically, a virtual northern blot shows expression inthe brain (adult and infant) and lung. In addition, PCR-based tissuescreening panels indicate expression in the infant brain andhippocampus. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) ornucleic acid expression.

Alternatively, a modulator for kinase nucleic acid expression can be asmall molecule or drug identified using the screening assays describedherein as long as the drug or small molecule inhibits the kinase nucleicacid expression in the cells and tissues that express the protein.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including infant and adult brain), lung, and hippocampus.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe kinase gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in kinase nucleic acid expression, and particularlyin qualitative changes that lead to pathology. The nucleic acidmolecules can be used to detect mutations in kinase genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the kinase gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the kinase gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a kinase protein.

Individuals carrying mutations in the kinase gene can be detected at thenucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the kinaseprotein of the present invention. SNPs were identified at nine differentnucleotide positions, including six positions outside the ORF. SNPsoutside the ORF may affect control/regulatory elements. As indicated bythe data presented in FIG. 3, the gene encoding the novel human kinaseprovided by the present invention is located on public BAC AC068701,which is known to be mapped to chromosome 3. Genomic DNA can be analyzeddirectly or can be amplified by using PCR prior to analysis. RNA or cDNAcan be used in the same way. In some uses, detection of the mutationinvolves the use of a probe/primer in a polymerase chain reaction (PCR)(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCRor RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see,e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa etal., PNAS 91:360-364 (1994)), the latter of which can be particularlyuseful for detecting point mutations in the gene (see Abravaya et al.,Nucleic Acids Res. 23:675-682 (1995)). This method can include the stepsof collecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

Alternatively, mutations in a kinase gene can be directly identified,for example, by alterations in restriction enzyme digestion patternsdetermined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantkinase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the kinase gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment. FIG. 3 provides information on SNPs that have been foundin the gene encoding the kinase protein of the present invention. SNPswere identified at nine different nucleotide positions, including sixpositions outside the ORF. SNPs outside the ORF may affectcontrol/regulatory elements.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol kinase gene expression in cells, tissues, and organisms. A DNAantisense nucleic acid molecule is designed to be complementary to aregion of the gene involved in transcription, preventing transcriptionand hence production of kinase protein. An antisense RNA or DNA nucleicacid molecule would hybridize to the mRNA and thus block translation ofmRNA into kinase protein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of kinase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired kinase nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the kinase protein, such as substratebinding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in kinase gene expression.Thus, recombinant cells, which include the patient's cells that havebeen engineered ex vivo and returned to the patient, are introduced intoan individual where the cells produce the desired kinase protein totreat the individual.

The invention also encompasses kits for detecting the presence of akinase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in humans in the brain (including infant andadult brain), lung, and hippocampus. Specifically, a virtual northernblot shows expression in the brain (adult and infant) and lung. Inaddition, PCR-based tissue screening panels indicate expression in theinfant brain and hippocampus. For example, the kit can comprise reagentssuch as a labeled or labelable nucleic acid or agent capable ofdetecting kinase nucleic acid in a biological sample; means fordetermining the amount of kinase nucleic acid in the sample; and meansfor comparing the amount of kinase nucleic acid in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectkinase protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application W095/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application W095/251 116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the kinase proteins/peptides of the present invention.In detail, such methods comprise incubating a test sample with one ormore nucleic acid molecules and assaying for binding of the nucleic acidmolecule with components within the test sample. Such assays willtypically involve arrays comprising many genes, at least one of which isa gene of the present invention and or alleles of the kinase gene of thepresent invention. FIG. 3 provides information on SNPs that have beenfound in the gene encoding the kinase protein of the present invention.SNPs were identified at nine different nucleotide positions, includingsix positions outside the ORF. SNPs outside the ORF may affectcontrol/regulatory elements.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified kinase gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

Vectors/host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby wellknown methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it maybe desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kuojan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection, DEAEdextran-mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection, lipofection, and other techniques such as thosefound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd,ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multitransmembrane domain containing proteins such as kinases,appropriate secretion signals are incorporated into the vector. Thesignal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with kinases, the protein can be isolated from the host cell bystandard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. The peptidecan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, anion orcationic exchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a kinaseprotein or peptide that can be further purified to produce desiredamounts of kinase protein or fragments. Thus, host cells containingexpression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe kinase protein or kinase protein fragments, such as those describedabove as well as other formats known in the art. Thus, a recombinanthost cell expressing a native kinase protein is useful for assayingcompounds that stimulate or inhibit kinase protein function.

Host cells are also useful for identifying kinase protein mutants inwhich these functions are affected. If the mutants naturally occur andgive rise to a pathology, host cells containing the mutations are usefulto assay compounds that have a desired effect on the mutant kinaseprotein (for example, stimulating or inhibiting function) which may notbe indicated by their effect on the native kinase protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a kinase proteinand identifying and evaluating modulators of kinase protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the kinase proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the kinase protein to particularcells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, kinase protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo kinaseprotein function, including substrate interaction, the effect ofspecific mutant kinase proteins on kinase protein function and substrateinteraction, and the effect of chimeric kinase proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more kinase proteinfunctions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

3 1 3124 DNA HUMAN 1 tccgcccccg gcccggggtg cgaatccggt gccggcaagcggctggcgat gctggaggtt 60 cgctagccga agcggctgca tctggcgccg cgtctgccccgcgtgctcgg agcggattct 120 gcccgccgtc cccggagccc tcggcgcccc gctgagcccgcgatcacttc ctccctgtga 180 ccaaccggcg ctgcaggtta gagcctggca atgccgtttgggtgtgtgac tctgggcgac 240 aagaagaact ataaccagcc atcggaggtg actgacagatatgatttggg acaggtcatc 300 aagactgagg agttttgtga aatcttccgg gccaaggacaagacgacagg caagctgcac 360 acctgcaaga agttccagaa gcgggacggc cgcaaggtgcggaaagctgc caagaacgag 420 ataggcatcc tcaagatggt gaagcatccc aacatcctacagctggtgga tgtgtttgtg 480 acccgcaagg agtactttat cttcctggag ctggccacggggagggaggt gtttgactgg 540 atcctggacc agggctacta ctcggagcga gacacaagcaacgtggtacg gcaagtcctg 600 gaggccgtgg cctatttgca ctcactcaag atcgtgcacaggaatctcaa gctggagaac 660 ctggtttact acaaccggct gaagaactcg aagattgtcatcagtgactt ccatctggct 720 aagctagaaa atggcctcat caaggagccc tgtgggacccccgagtatct ggccccagag 780 gtggtaggcc ggcagcggta tggacgccct gtggactgctgggccattgg agtcatcatg 840 tacatcctgc tttcaggcaa tccacctttc tatgaggaggtggaagaaga tgattatgag 900 aaccatgata agaatctctt ccgcaagatc ctggctggtgactatgagtt tgactctcca 960 tattgggatg atatttcgca ggcagccaaa gacctggtcacaaggctgat ggaggtggag 1020 caagaccagc ggatcactgc agaagaggcc atctcccatgagtggatttc tggcaatgct 1080 gcttctgata agaacatcaa ggatggtgtc tgtgcccagattgaaaagaa ctttgccagg 1140 gccaagtgga agaaggctgt ccgagtgacc accctcatgaaacggctccg ggcaccagag 1200 cagtccagca cggctgcagc ccagtcggcc tcagccacagacactgccac ccccggggct 1260 gcaggtgggg ccacagctgc agctgcgagt ggagctacctcagcccctga gggtgatgct 1320 gctcgtgctg caaagagtga taatgtggcc cccgcagaccgtagtgccac cccagccaca 1380 gatggaagtg ccaccccagc cactgatggc agtgtcaccccagccaccga tggaagcatc 1440 actccagcca ctgatgggag tgtcacccca gtcactgacaggagcgctac tccagccact 1500 gatgggagag ccacaccagc cacagaagag agcactgtgcccaccaccca aagcagtgcc 1560 atgctggcca ccaaggcagc tgccacccct gagccggctatggcccagcc ggacagcaca 1620 gccccagagg gcgccacagg ccaggctcca ccctctagtaaaggggaaga ggctgctggt 1680 tatgcccagg agtctcaaag ggaggaggcc agctgagtaggcagcctggt gagggggggc 1740 aggggatggg caggagggtg ggagagtgga tgaggggcttctcactgtac atagagtcac 1800 tggcatgatg ccctcgctcc cccatgcccc cacatcccagtggggcataa ctaggggtca 1860 cgggagagca gtctcgtctc ctgtgtgtat gtgtgtgagtggtgggcagg ccagtggcag 1920 ggccggcccc agcccctgca tggattcctt gtggcttttctgtcttttgc tagcttcacc 1980 agtttctgtt ccttgtggga tgctgctcta gggatactcagggggctcct gctctccttc 2040 cccttccctt cttgcctcac cattccccta ggcaggccctgcaggtccca cactctccca 2100 ggccctaaac ttgggcggcc ttgccctgag agctggtcctccagcgaggc cctgtcagcg 2160 gtcttaggct cctgcacatg aaggtgtgtg cctgtggtgtgtgggctgct ctaggagcag 2220 atacaggctg gtatagagga tgcagaaagg tagggcagtatgtttaagtc cagacttggc 2280 acatggctag ggatactgct cactagctgt ggaggtcctcaggagtggag agaatgagta 2340 ggagggcaga agcttccatt tttgtccttc ctaagaccctgttatttgtg ttatttcctg 2400 cctttccgag tcctgcagtg ggctgccctg taccctgaacctcatgagcc tctaagggaa 2460 aggaggaaca attaggacgt ggcaatgaga cctggcagggcagagtacaa gcccagcacc 2520 cagtgtccca gccttactgg gtccttaccc tgggccaaacagggagggct gatacctcct 2580 tgctcttcct agatgcccac ctcctacaat ctcagcccacaagtcctctc caccctaggg 2640 ggcttgctgc atggcaataa ctcataatct gatttggaggtttgcccttt acaggggcag 2700 attttctgct cagttcaaca atgaaatgaa gaggaactccctctttctac agctcacttc 2760 tatcagaggc ccaggtgcct cagagccaca ttgagttgctttttctggga tgaggaagta 2820 gggttaaact ccccagtttc ctgagggagg ctcctgacaggtgccctttg tcagacccta 2880 ccacagcctg gataggcagc cacattggtc ctcgcccttgctcggcactc cgtggtggtc 2940 ctgcccttct ccctgcatgc ctgtgggtct gctctggtgtgtgaaggtcg gtgggttaac 3000 tgtgtgccta ctgaacctgg caaataaaca tcaccctgcaaagccaaaaa aaaaaaaaaa 3060 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 3120 aaaa 3124 2 501 PRT HUMAN 2 Met Pro Phe GlyCys Val Thr Leu Gly Asp Lys Lys Asn Tyr Asn Gln 1 5 10 15 Pro Ser GluVal Thr Asp Arg Tyr Asp Leu Gly Gln Val Ile Lys Thr 20 25 30 Glu Glu PheCys Glu Ile Phe Arg Ala Lys Asp Lys Thr Thr Gly Lys 35 40 45 Leu His ThrCys Lys Lys Phe Gln Lys Arg Asp Gly Arg Lys Val Arg 50 55 60 Lys Ala AlaLys Asn Glu Ile Gly Ile Leu Lys Met Val Lys His Pro 65 70 75 80 Asn IleLeu Gln Leu Val Asp Val Phe Val Thr Arg Lys Glu Tyr Phe 85 90 95 Ile PheLeu Glu Leu Ala Thr Gly Arg Glu Val Phe Asp Trp Ile Leu 100 105 110 AspGln Gly Tyr Tyr Ser Glu Arg Asp Thr Ser Asn Val Val Arg Gln 115 120 125Val Leu Glu Ala Val Ala Tyr Leu His Ser Leu Lys Ile Val His Arg 130 135140 Asn Leu Lys Leu Glu Asn Leu Val Tyr Tyr Asn Arg Leu Lys Asn Ser 145150 155 160 Lys Ile Val Ile Ser Asp Phe His Leu Ala Lys Leu Glu Asn GlyLeu 165 170 175 Ile Lys Glu Pro Cys Gly Thr Pro Glu Tyr Leu Ala Pro GluVal Val 180 185 190 Gly Arg Gln Arg Tyr Gly Arg Pro Val Asp Cys Trp AlaIle Gly Val 195 200 205 Ile Met Tyr Ile Leu Leu Ser Gly Asn Pro Pro PheTyr Glu Glu Val 210 215 220 Glu Glu Asp Asp Tyr Glu Asn His Asp Lys AsnLeu Phe Arg Lys Ile 225 230 235 240 Leu Ala Gly Asp Tyr Glu Phe Asp SerPro Tyr Trp Asp Asp Ile Ser 245 250 255 Gln Ala Ala Lys Asp Leu Val ThrArg Leu Met Glu Val Glu Gln Asp 260 265 270 Gln Arg Ile Thr Ala Glu GluAla Ile Ser His Glu Trp Ile Ser Gly 275 280 285 Asn Ala Ala Ser Asp LysAsn Ile Lys Asp Gly Val Cys Ala Gln Ile 290 295 300 Glu Lys Asn Phe AlaArg Ala Lys Trp Lys Lys Ala Val Arg Val Thr 305 310 315 320 Thr Leu MetLys Arg Leu Arg Ala Pro Glu Gln Ser Ser Thr Ala Ala 325 330 335 Ala GlnSer Ala Ser Ala Thr Asp Thr Ala Thr Pro Gly Ala Ala Gly 340 345 350 GlyAla Thr Ala Ala Ala Ala Ser Gly Ala Thr Ser Ala Pro Glu Gly 355 360 365Asp Ala Ala Arg Ala Ala Lys Ser Asp Asn Val Ala Pro Ala Asp Arg 370 375380 Ser Ala Thr Pro Ala Thr Asp Gly Ser Ala Thr Pro Ala Thr Asp Gly 385390 395 400 Ser Val Thr Pro Ala Thr Asp Gly Ser Ile Thr Pro Ala Thr AspGly 405 410 415 Ser Val Thr Pro Val Thr Asp Arg Ser Ala Thr Pro Ala ThrAsp Gly 420 425 430 Arg Ala Thr Pro Ala Thr Glu Glu Ser Thr Val Pro ThrThr Gln Ser 435 440 445 Ser Ala Met Leu Ala Thr Lys Ala Ala Ala Thr ProGlu Pro Ala Met 450 455 460 Ala Gln Pro Asp Ser Thr Ala Pro Glu Gly AlaThr Gly Gln Ala Pro 465 470 475 480 Pro Ser Ser Lys Gly Glu Glu Ala AlaGly Tyr Ala Gln Glu Ser Gln 485 490 495 Arg Glu Glu Ala Ser 500 3 7542DNA HUMAN 3 aatgaaatcc tgtatctaca aaaaaatttt taaaaattag ccagttgtggtggtgtgcac 60 ctatggtcca gtcctagcta ctcaggaggc tgaggtggga ggatcgcttgagcccaggag 120 gttgaggctg gaatgagctg tgatcattcc actgcattcc agcctgggtgccactgcatt 180 ccagcctggg tgacagaaca gctgggcatg gtgcgggtgc ctgtaatcccagctgcggtg 240 ggaggatcgt ttgagttcag gagctctggg ctgcagtgcg ctatgccagttgggtgtcta 300 cactaagttt ggcattagta tggtgacttc ctgggagcta gagctcatcaggttgcctaa 360 aggaggggtg aagcaaccaa ggtcagagac gaagcaggtc aaaactcctgtgctgatcag 420 tagtgggctt gtgcccatga atagccactg cactccagcc tgggcaacatagtgagactt 480 gtctctaaaa aaagaaagaa agaaagaaaa gaaaagaaaa gaaataggtcttccagaatc 540 ccccccatgt gtcctgaatc aatcataacc cctccttttc tcttagaagggaccattatc 600 tgactctgct tatttcctag cttttcttta tagctctact gctgtgtttgtatccccctt 660 acgggtttgt tctgcctgtt tgaactctat caatagactc atactatatgtattcttttg 720 tgtctttttg cacttaacac tgtgtaagag tcaccaggtg gttgggtatagctgtagctc 780 ctttgtttcc attgctatat aatattccat tgtgtgactc taccacaatacatcctgttg 840 gtggtggtat ttggtctgtt ttcaattttt ggcgattatg tatatggctactatgagcat 900 tgtatgactg tcctgatgca cgaatatgtg cacttctgta ggcacgtatttaggagtgca 960 attgctggtt catagggtat gtttgtttga ctttagtggt tttaccaaactgctttacaa 1020 aggggagttt gccctctcct agcagtgctc acgttcttca tatatccactaacacttgtt 1080 agctctagac cttcagagat gactagcagg tgggtgggtg gttatagcccccttcatctg 1140 taaaatagct gaaatttaaa taaactgttt gataagcctt tttttgaatgtatagaatac 1200 aaaactattc ccttatatat cagcttaata cattttttca tatgtaggctgttgcacttc 1260 aggcagccaa gctgagtagc tggatgtcta gccagaaaag caggggtaggatctaagcct 1320 gcctatcctc tagcccctcc tggtatggtc tctgccccta gcttggcctgagccaggttg 1380 gggtctccca aaagcagccc ctatgttgtg gggaagtgga gagtagtacagctaagccag 1440 accccattgt gcccgcaggt tagagcctgg caatgccgtt tgggtgtgtgactctgggcg 1500 acaagaagaa ctataaccag ccatcggagg tgactgacag atatgatttgggacaggtca 1560 tcaagacgtg agtgccgggc ctgtcaggca aggctgggtg ggctggtgagcaggggctag 1620 ggaagtggca gtggagtgca gctgatgcta aggccaggcc tgagtgggtgcctgtcggct 1680 gcagtgagga gttttgtgaa atcttccggg ccaaggacaa gacgacaggcaagctgcaca 1740 cctgcaagaa gttccagaag cgggacggcc gcaaggtgcg gaaagctgccaagaacgaga 1800 taggcatcct caagatgtga gtttgaggcc tgggattggg gcggggtgggaggcagcagg 1860 gaagggctta gagggcaagt ggcctcaggc agtcccagcc tctggccaattagtactgct 1920 gtggccactg tggtgaccac agcctctccc aggggttcat ccagccctaactactggctc 1980 ccctccctgt cgccagggtg aagcatccca acatcctaca gctggtggatgtgtttgtga 2040 cccgcaagga gtactttatc ttcctggagc tgtgagtgtg ggtctggggacccaagattc 2100 cccagcgccc agggctttca cctgtcccac cctctgcagc taaggaaaccccctttctgg 2160 acccttggcg tcccaggtcc ctgtgccttg ctcagccagc tgcctggcgtaggcagctca 2220 cccaagtgct cctgccctac ccctgattct gcccacgcca ggctcaggggctggtgtccc 2280 tcagggccac ggggagggag gtgtttgact ggatcctgga ccagggctactactcggagc 2340 gagacacaag caacgtggta cggcaagtcc tggaggccgt ggcctatttgcactcactca 2400 agatcgtgca caggaatctc aaggtgaggg cagagccaga gtcaaagggccagttggcag 2460 ctgggctggt gctggggtgg gcagcacctc agggccttgg tctagcctccaaggtcctca 2520 tggtgtcttc tgctcaccca catgctattc tcacaccaca gctggagaacctggtttact 2580 acaaccggct gaagaactcg aagattgtca tcagtgactt ccatctggctaagctagaaa 2640 atggcctcat caaggagccc tgtgggaccc ccgagtatct gggcaagcagggggtggggc 2700 aggggcaggg ggataatggg gggcagcctt cagggagctg ctctgggcagggggaaaatg 2760 tgctcatctc aggaagctgg tgtggatggt actggacctg gctaggcctgatactgacca 2820 gcagatgggc gctgtgtttg tagccccaga ggtggtaggc cggcagcggtatggacgccc 2880 tgtggactgc tgggccattg gagtcatcat gtacatcctg tgagtggacagatggacaag 2940 caggcttgca gtcagatggg gtgggggcat gtgtctgtgg cctttctgtgtgacccttcc 3000 cccatgcagg ctttcaggca acccaccttt ctatgaggag gtggaagaagatgattatga 3060 gaaccatgat aagaatctct tccgcaagat cctggctggt gactatgagtttgactctcc 3120 atattgggat gatatttcgc aggcaggtga ggaggtgcct gcccagcacttggtagaacg 3180 caagggagtg gggagggtgt cctgctctca tcttcctctg tggtattctgcttctgtccc 3240 ccagcccatt tatcacctct agacattgag atggggtgcc catgactatcccctccttgg 3300 ttttgtccac agccaaagac ctggtcacaa ggctgatgga ggtggagcaagaccagcgga 3360 tcactgcaga agaggccatc tcccatgagt ggtgagcagg gtccaggggagggtgggaga 3420 ggccagggtc cccctcacac ccagcagccc catctttgag gcctagaaggggtgcaggta 3480 cctagagctc agggcagccc agaggctctg cctggtagtc cctggatgccacatagaagg 3540 gctgagcaga gtctccacaa aggctcattc tctcagatcc agacacacttgcaccctttc 3600 caggatttct ggcaatgctg cttctgataa gaacatcaag gatggtgtctgtgcccagat 3660 tgaaaagaac tttgccaggg ccaagtggaa ggtaaggctt ggataactcccttcttggct 3720 ctatcccaag tattgcttct ggcactcagt ttcttgcctg catcacacccctgcagccac 3780 acacaagctt ttctcaggcc atcaggtgtg ggtgtagcca ctgtgattaccttaagtagg 3840 agtgctgagc agctgagtac ttggccctgg ggtggatgga ggtggccgggggtactatgg 3900 aaggagtttg gtttggtcac tgccaagcaa tggatccagc aagcctcacccttagccttt 3960 ctccactgtg ctcctttcag aaggctgtcc gagtgaccac cctcatgaaacggctccggg 4020 caccagagca gtccagcacg gctgcagccc agtcggcctc agccacagacactgccaccc 4080 ccggggctgc aggtggggcc acagctgcag ctgcgagtgg agctacctcagcccctgagg 4140 gtgatgctgc tcgtgctgca aagagtgata atgtggcccc cgcagaccgtagtgccaccc 4200 cagccacaga tggaagtgcc accccagcca ctgatggcag tgtcaccccagccaccgatg 4260 gaagcatcac tccagccact gatgggagtg tcaccccagc cactgacaggagcgctactc 4320 cagccactga tgggagagcc acaccagcca cagaagagag cactgtgcccaccacccaaa 4380 gcagtgccat gctggccacc aaggcagctg ccacccctga gccggctatggcccagccgg 4440 acagcacagc cccagagggc gccacaggcc aggctccacc ctctagtaaaggggaagagg 4500 ctgctggtta tgcccaggag tctcaaaggg aggaggccag ctgagtaggcagcctggtga 4560 gggggggcag gggatgggca ggagggtggg agagtggatg aggggcttctcactgtacat 4620 agagtcactg gcatgatgcc ctcgctcccc catgccccca catcccagtggggcataact 4680 aggggtcacg ggagagcagt ctcgtctcct gtgtgtatgt gtgtgagtggtgggcaggcc 4740 agtggcaggg ccggccccag cccctgcatg gattccttgt ggcttttctgtcttttgcta 4800 gcttcaccag tttctgttcc ttgtgggatg ctgctctagg gatactcagggggctcctgc 4860 tctccttccc cttcccttct tgcctcacca ttcccctagg caggccctgcaggtcccaca 4920 ctctcccagg ccctaaactt gggcggcctt gccctgagag ctggtcctccagcgaggccc 4980 tgtcagcggt cttaggctcc tgcacatgaa ggtgtgtgcc tgtggtgtgtgggctgctct 5040 aggagcagat acaggctggt atagaggatg cagaaaggta gggcagtatgtttaagtcca 5100 gacttggcac atggctaggg atactgctca ctagctgtgg aggtcctcaggagtggagag 5160 aatgagtagg agggcagaag cttccatttt tgtccttcct aagaccctgttatttgtgtt 5220 atttcctgcc tttccgagtc ctgcagtggg ctgccctgta ccctgaacctcatgagcctc 5280 taagggaaag gaggaacaat taggacgtgg caatgagacc tggcagggcagagtacaagc 5340 ccagcaccca gtgtcccagc cttaatgggt ccttaccatg ggccaaacagggagggctga 5400 tacctccttg ctcttcctag atgcccacct cctacaatct cagcccacaagtcctctcca 5460 cctagggggc ttgctgcatg gcaataactc ataatttgat ttggaggtttgccctttaca 5520 ggggcagatt ttctgctcag ttcaacaatg aaatgaagag gaactccctctttctacagc 5580 tcacttctat cagaggccca ggtgcctcag agccacattg agttgctttttctgggatga 5640 ggaagtaggg ttaaactccc cagtttcctg agggaggctc ctgacaggtgccctttgtca 5700 gaccctacca cagcctggat aggcagccac attggtcctc gcccttgctcggcactccgt 5760 ggtggtcctg cccttctccc tgcatgcctg tgggtctgct ctggtgtgtgaaggtcggtg 5820 ggttaactgt gtgcctactg aacctggcaa ataaacatca ccctgcaaagcctctggcca 5880 ccctctcgcc tttgcttcct ctgtcctact ggggaggagc ccctggaaggcagtggggaa 5940 gggagaggct gggagcaggt tcacaagtag tggctgggag tagtagtgaacagtgccctg 6000 tggatttctt gggctgaggg tgaagatcat cctcaccaca gtgtattcctcaatggtggt 6060 ggtggtgggg gtcctctgaa ctctagaaat ccatgtgagt agtgggtcttgggctgcatt 6120 tggaccaagg aatactccct gggctgagag ctcaagtaag ggctgctgtttttgactcct 6180 gttgaccgag gagccttact cttgatttag gcatagagct ggaatctacctgggccagcc 6240 agcgcaagga cagaccctgc tcatagccaa agatagcttc cctgagctagggagagggtg 6300 tagggtgagc aatgcacaaa gattctatgc tgctctgtgt tgattccttctttctgtgaa 6360 cacctgtgct ttccacctat gccccaaatg aaatctcact ctgggtctatttcagctgga 6420 gcagatcagc tttatattat gggactatgc acagggtttc ttctgggtagagggagtaac 6480 actgccacag atactaagcc taaaaaccca aaagttgact tactccagcccaggtgggcc 6540 tgggtgaact caccagggat gctgctctct gggaggacct caaagataacctcctcaagg 6600 ataacttctc tgatgtggga ataataaaaa ggatttgtta gtctctagtctttgaagcat 6660 ctgggcctcc aaaattccaa ccctactaca gtctcaagca taactgatcctcctgagagc 6720 taagagctaa agtcagggta ctggccctag tgtctccaaa ctcaaggatagtcatatctt 6780 acttggatga agccaaggtt ggcctggggt gacctgaggg actggggcctatgtgtccat 6840 ctttctagaa tcgcccggaa ctgaggactg agagggtagg gtattgtggtctatgggaac 6900 tacagaccat agcgcatggt ccatcactgt ccagagccca agggacccatcgggaccatt 6960 gttctattcc cttgcggctg cggttttctc tgccttggcc cagcctgcctcgcgccagct 7020 ctggcatggg accacaatgc gctgccttgc cctggaaaag ggtccagctggcagcctcgc 7080 tgctccccgc cttgatataa tgctgcgctc tcaagtggca ggtcgagtctccccagagga 7140 cgcctgagag cgaccgcgct ccgcccagga ttctctacgg cctcggggccgctaagcccc 7200 gcccatagtt ccgcccatcc gagaatttgt ccaatccagg cgaagtattcacgctccgcc 7260 caaagttttg tgttgtcact tccggcccgc tccaggaagt cgtgctgcggagccaaattt 7320 gaagcaagcg gaggcgcggg ggcgcgtcta cgaagccgga cctgtagcagtttctttggc 7380 tgcctgggcc ccttgagtcc agccatcatg cctatccgtg ctctgtgcactatctgctcc 7440 gacttcttcg atcactcccg cgacgtggcc gccatccact gcggccacaccttccacttg 7500 cagtggtgag tgactgcagt gttggggccc ggtgtttgcc cg 7542

That which is claimed is:
 1. An isolated nucleic acid moleculeconsisting of a nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence that encodes a protein comprising theamino acid sequence of SEQ ID NO:2; (b) a nucleotide sequence consistingof SEQ ID NO:1; (c) a nucleotide sequence consisting of SEQ ID NO:3; and(d) a nucleotide sequence that is completely complementary to anucleotide sequence of (a)-(c).
 2. A nucleic acid vector comprising anucleic acid molecule of claim
 1. 3. A host cell containing the vectorof claim
 2. 4. A process for producing a polypeptide comprisingculturing the host cell of claim 3 under conditions sufficient for theproduction of mid polypeptide, and recovering said polypeptide from thehost cell culture.
 5. An isolated polynucleotide consisting of anucleotide sequence set forth in SEQ ID NO:1.
 6. An isolatedpolynucleotide consisting of a nucleotide sequence set forth in SEQ IDNO:3.
 7. A vector according to claim 2, wherein said vector is selectedfrom the group consisting of a plasmid, virus, and bacteriophage.
 8. Avector according to claim 2, wherein said isolated nucleic acid moleculeis inserted into said vector in proper orientation and correct readingframe such that the protein of SEQ ID NO:2 may be expressed by a celltransformed with said vector.
 9. A vector according to claim 8, whereinsaid isolated nucleic acid molecule is operatively linked to a promotersequence.